Quantitative Real-time PCR Assay For The Detection Of Propionibacterium acnes

Abstract

Purpose: : Propionibacterium acnes (P. acnes) is a common cause of delayed-onset postoperative endophthalmitis and a potential cause of microbial keratitis. It has a very slow growth rate using conventional culture method and culture negative rate is extremely high. We have developed a quantitative real-time PCR assay to detect P. acnes in ocular infections.

Methods: : In order to identify a gene target specific for P. acnes but not for different Propionibacterium spp. or other organisms commonly found in ocular infections, PCR assays targeting the seven housekeeping genes used in the P. acnes multilocus sequence typing (MLST) scheme was carried out on five different Propionibacterium spp. and four other microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and coagulase negative Staphylococcus). PCR products were determined by gel electrophoresis on agarose gels. First round of PCR was then carried out using conventional PCR targeting the selected housekeeping gene and the PCR product was transferred to a second round real-time PCR in a LightCycler®. This was undertaken on serial dilutions of P. acnes genomic DNA (0.1pg/ml to 1ng/ml) to detect sensitivity of the assay.

Results: : Three genes, gmk, guaA and recA were found to be specific for P. acnes. Gmk was the smallest PCR product using nested primers and was chosen as the rapid diagnostic target as this would allow more rapid PCR cycling. Melt-curve analysis of the real-time PCR reaction shows that the limit of detection of the assay was 0.1pg/ml. Amplification plot generated from the serial dilution data allows quantification of unknown amounts of starting DNA material. The total assay time including handling can be completed within 2 hours 30 minutes.

Conclusions: : The real-time PCR method described here to detect P. acnes is much more sensitive and rapid than the conventional culture method employed in most clinical laboratories, which can take up to ten days. It is also more specific and less labour intensive than PCR diagnostic assays utilising universal primers that target 16S rRNA genes.

Keywords: endophthalmitis • microbial pathogenesis: experimental studies • clinical laboratory testing